The detection and quantitative determination of hydrogen peroxide and compounds yielding hydrogen peroxide as a result of chemical or enzymatic reactions are of importance in many areas. For example, they are important in the detection of hydrogen peroxide produced in the enzymatic assay of chemical or biological substances (sometimes called analytes) such as glucose, cholesterol, uric acid, triglycerides, creatine kinase, etc. in the presence of oxygen. The quantity of analyte present in a specimen sample is determinable from the amount of hydrogen peroxide produced and detected.
Known compositions for detecting or quantifying hydrogen peroxide in such assays generally comprise a substance having peroxidative activity, e.g. peroxidase, and a material which undergoes a detectable change (e.g. a color change) in the presence of hydrogen peroxide and the peroxidative substance. Various materials which undergo such a detectable change include monoamines, diamines, phenols, leuco dyes and other known dyes or dye formers. Dye-providing materials also useful in such assays include triarylimidazoles as described, for example, in U.S. Pat. No. 4,089,747 (issued May 16, 1978 to Bruschi).
Triarylmethane dyes and their leuco precursors are also known as commercially useful compounds. Triarylmethane leuco dyes, for example, are known as useful indicators of hydrogen peroxide. Examples of such leuco dyes include the following compounds: ##STR2## However, it has been observed that such leuco dyes readily oxidize in air or in aqueous solutions containing a substance having peroxidative activity (see e.g. Example 1 hereinbelow). Hence, this instability renders them unsuitable for analytical determinations, and especially for dry assays where the analytical composition is stored for a period of time prior to use.
Although other dye-providing materials have the desired stability and are, in general, useful as indicators for hydrogen peroxide determination, there are instances when the concentration of hydrogen peroxide to be analyzed is too low to produce sufficient detectable color from such indicators. In some instances, this shortcoming can be overcome by using increased amounts of indicator. However, where the analyte concentration is initially low or high dilution of the test sample is required, such indicators may still be deficient because they still provide insufficient detectable color in such instances.
Such problems of instability and low analyte concentration are particularly acute when analyte determination is attempted with a dry analytical element, e.g. with the commercially successful elements described in U.S. Pat. No. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al). In such instances, the indicator or reagent layer present in such elements is necessarily very thin, and the dye concentration is relatively low. Hence, the density of the color formed can be rather low even with high analyte concentrations. However, it would be desirable to use such elements for very low analyte concentrations.
Hence, there is a continuing need in the art for dye-providing materials which are stable to oxidation and which can be used to detect low concentrations of hydrogen peroxide or of analytes which react to produce same.